Functional significance may underlie the taxonomic utility of single amino acid substitutions in conserved proteins

We hypothesized that some amino acid substitutions in conserved proteins that are strongly fixed by critical functional roles would show lineage-specific distributions. As an example of an archetypal conserved eukaryotic protein we considered the active site of ß-tubulin. Our analysis identified one amino acid substitution—ß-tubulin F224—which was highly lineage specific. Investigation of ß-tubulin for other phylogenetically restricted amino acids identified several with apparent specificity for well-defined phylogenetic groups. Intriguingly, none showed specificity for “supergroups” other than the unikonts. To understand why, we analysed the ß-tubulin Neighbor-Net and demonstrated a fundamental division between core ß-tubulins (plant-like) and divergent ß-tubulins (animal and fungal). F224 was almost completely restricted to the core ß-tubulins, while divergent ß-tubulins possessed Y224. Thus, our specific example offers insight into the restrictions associated with the co-evolution of ß-tubulin during the radiation of eukaryotes, underlining a fundamental dichotomy between F-type, core ß-tubulins and Y-type, divergent ß-tubulins. More broadly our study provides proof of principle for the taxonomic utility of critical amino acids in the active sites of conserved proteins

Microtubules in Bacteria: Ancient Tubulins Build a Five-Protofilament Homolog of the Eukaryotic Cytoskeleton

Microtubules play crucial roles in cytokinesis, transport, and motility, and are therefore superb targets for anti-cancer drugs. All tubulins evolved from a common ancestor they share with the distantly related bacterial cell division protein FtsZ, but while eukaryotic tubulins evolved into highly conserved microtubule-forming heterodimers, bacterial FtsZ presumably continued to function as single homopolymeric protofilaments as it does today. Microtubules have not previously been found in bacteria, and we lack insight into their evolution from the tubulin/FtsZ ancestor. Using electron cryomicroscopy, here we show that the tubulin homologs BtubA and BtubB form microtubules in bacteria and suggest these be referred to as “bacterial microtubules” (bMTs). bMTs share important features with their eukaryotic counterparts, such as straight protofilaments and similar protofilament interactions. bMTs are composed of only five protofilaments, however, instead of the 13 typical in eukaryotes. These and other results suggest that rather than being derived from modern eukaryotic tubulin, BtubA and BtubB arose from early tubulin intermediates that formed small microtubules. Since we show that bacterial microtubules can be produced in abundance in vitro without chaperones, they should be useful tools for tubulin research and drug screening

Análise de timol em cera de abelha por micro-extracção em fase sólida (SPME)

A aplicação contínua de acaricídas lipofílicos sintéticos no tratamento das abelhas conduz a uma acumulação que depende da frequência, lipofilicidade e quantidade de princípio activo utilizada. Este efeito é mais acentuado na cera de abelha que no mel, no entanto, e porque a persistência destes resíduos é elevada, provoca o aparecimento de resistências e a perda do seu efeito acaricida.[1] Esta razão levou à pesquisa de outros compostos alternativos não tóxicos e não persistentes, com efeito sobre o ácaro das abelhas, Varroa Jacobsoni. Entre estes compostos encontra-se o timol, um composto fenólico, volátil, presente no tomilho. Dos diversos componentes dos óleos essenciais este é sem dúvida o que demonstrou maior efeito acaricida, utilizando-se no tratamento das abelhas directamente ou como componente de diversas formulações.[2] Em Portugal, foi introduzido muito recentemente sob a forma comercial de APIGUARD: um gel, à base de timol, que controla termicamente a libertação do princípio activo. O controlo dos resíduos de timol na cera de abelha e no mel é assim um desafio actual quer do ponto de vista sanitário quer de qualidade alimentar. A micro-extracção em fase sólida (SPME) é uma técnica de preparação de amostras que se baseia na sorção de analítos no revestimento de uma fibra de sílica fundida e posterior desorção térmica no injector de um cromatógrafo em fase gasosa (GC). Para além de combinar num único processo etapas de extracção, purificação e concentração dos analitos, a técnica de SPME apresenta uma série de vantagens relativamente às técnicas de extracção convencionais, como a extracção líquido-líquido e extracção em fase sólida, nomeadamente a sua relativa simplicidade e rapidez, reduzido custo e não utilização de solventes para a extracção de analitos, para além de permitir a extracção por imersão directa na amostra gasosa ou líquida e extracção por amostragem do espaço-de-cabeça da amostra líquida ou sólida.[3] Ao contrário das técnicas tradicionais, que permitem uma extracção quantitativa dos analitos, a técnica de SPME baseia-se num equilíbrio de partição do analito. Esta particularidade torna a técnica de SPME bastante sensível a parâmetros experimentais que possam afectar os coeficientes de partição dos analitos e, consequentemente, a sensibilidade e reprodutibilidade dos resultados.[4] O objectivo deste trabalho é o desenvolvimento de uma metodologia para a análise de timol em ceras contaminadas, utilizando como padrão interno a benzofenona. Em primeiro lugar, procedeu-se à optimização da técnica através da determinação da quantidade de cera, temperatura de análise e período de contacto da fibra com o espaço-de-cabeça da amostra mais adequados para o caso em estudo. Numa segunda fase, procedeu-se à análise de diversas lâminas de cera contaminadas propositadamente com timol e sujeitas a diferentes condições de armazenamento: em frio, ao ar e em estufa. Finalmente, procedeu-se à construção da curva de calibração e quantificação do timol presente nas diversas amostras de cera analisadas. Considerando-se os resultados, para os níveis de contaminação avaliados, as condições analíticas mais adequadas ocorrem com a utilização de 1 g de cera, mantendo-se a fibra em contacto com o espaço-de-cabeça durante 40 minutos a uma temperatura de 60 ºC. Nestas condições experimentais foi possível obter uma boa correlação linear (r2=0,990) no intervalo de concentrações [3,5-14 mg/g]. A quantidade de timol encontrada nas amostras é significativamente inferior à colocada durante o processo de fabrico das lâminas, pelo que o processo de conservação não é o mais adequado, sendo evidente uma menor quantidade de timol quando a lâmina de cera é colocada na estufa

Pydna: a simulation and documentation tool for DNA assembly strategies using python

Background: Recent advances in synthetic biology have provided tools to efficiently construct complex DNA molecules which are an important part of many molecular biology and biotechnology projects. The planning of such constructs has traditionally been done manually using a DNA sequence editor which becomes error-prone as scale and complexity of the construction increase. A human-readable formal description of cloning and assembly strategies, which also allows for automatic computer simulation and verification, would therefore be a valuable tool.Results: We have developed pydna, an extensible, free and open source Python library for simulating basic molecular biology DNA unit operations such as restriction digestion, ligation, PCR, primer design, Gibson assembly and homologous recombination. A cloning strategy expressed as a pydna script provides a description that is complete, unambiguous and stable. Execution of the script automatically yields the sequence of the final molecule(s) and that of any intermediate constructs. Pydna has been designed to be understandable for biologists with limited programming skills by providing interfaces that are semantically similar to the description of molecular biology unit operations found in literature.Conclusions: Pydna simplifies both the planning and sharing of cloning strategies and is especially useful for complex or combinatorial DNA molecule construction. An important difference compared to existing tools with similar goals is the use of Python instead of a specifically constructed language, providing a simulation environment that is more flexible and extensible by the user.Thanks to Dr. Aric Hagberg Los Alamos National Laboratory, U.S.A and Sergio Simoes, Universidade de Sao Paulo, Brasil for help with NetworkX and graph theory in general. Thanks to Henrik Bengtsson, Dept of Epidemiology & Biostatistics, University of California San Francisco, U.S.A. for critical reading of the manuscript. Thanks to the 2013 Bioinformatics 6605 N4 students A. Coelho, A. Faria, A. Neves D. Yelshyna and E. Costa for testing. This work was supported by the Fundacao para a Ciencia e Tecnologia (FCT) [PTDC/AAC-AMB/120940/2010, EXPL/BBB-BIO/1772/2013]; and the FEDER POFC-COMPETE [PEst-C/BIA/UI4050/2011]. FA and GR were supported by FCT fellowships [SFRH/BD/80934/2011 and SFRH/BD/42565/2007, respectively].info:eu-repo/semantics/publishedVersio

TRÁFICO DE PESSOAS PARA FINS DE EXPLORAÇÃO SEXUAL E SEU ENFRENTAMENTO SOB A ÓTICA INTERNACIONAL E NACIONAL

O presente artigo discorre sobre o enfrentamento do crime de tráfico humano, com enfoque na sua ocorrência para fins de exploração sexual. Por intermédio de análise documental, busca-se analisar a evolução da legislação pertinente, bem como sua eficácia na prevenção e repressão do delito em voga. Observado o contexto histórico em que o tráfico humano se encontra inserido, bem como sua conceituação e formas de incidência em uma realidade fática, são exploradas as circunstâncias amplamente favoráveis ao persistente crescimento do crime no âmago da sociedade. Destarte, será possível constatar que muito embora tenha o Brasil logrado avanços significativos com o advento da Lei 13.344/2016 sobre o tráfico humano, o enfrentamento no país ainda se dá de forma insuficiente, uma vez que pouco é abordado no cotidiano, permanecendo o crime, assim, carente de maior atenção político-criminal, o que dificulta sua prevenção e repressão

Enteric Infection with Citrobacter rodentium Induces Coagulative Liver Necrosis and Hepatic Inflammation Prior to Peak Infection and Colonic Disease

Acute and chronic forms of inflammation are known to affect liver responses and susceptibility to disease and injury. Furthermore, intestinal microbiota has been shown critical in mediating inflammatory host responses in various animal models. Using C. rodentium, a known enteric bacterial pathogen, we examined liver responses to gastrointestinal infection at various stages of disease pathogenesis. For the first time, to our knowledge, we show distinct liver pathology associated with enteric infection with C. rodentium in C57BL/6 mice, characterized by increased inflammation and hepatitis index scores as well as prominent periportal hepatocellular coagulative necrosis indicative of thrombotic ischemic injury in a subset of animals during the early course of C. rodentium pathogenesis. Histologic changes in the liver correlated with serum elevation of liver transaminases, systemic and liver resident cytokines, as well as signal transduction changes prior to peak bacterial colonization and colonic disease. C. rodentium infection in C57BL/6 mice provides a potentially useful model to study acute liver injury and inflammatory stress under conditions of gastrointestinal infection analogous to enteropathogenic E. coli infection in humans.United States. Army Research Office (Institute for Soldier Nanotechnology grant 6915539 (SRT))National Institutes of Health (U.S.) (Grant P01 CA026731)National Institutes of Health (U.S.) (Grant P30 ES02109)National Institutes of Health (U.S.) (Toxicology Training grant ES-070220

A novel murine infection model for Shiga toxin-producing Escherichia coli

Enterohemorrhagic E. coli (EHEC) is an important subset of Shiga toxin-producing (Stx-producing) E. coli (STEC), pathogens that have been implicated in outbreaks of food-borne illness and can cause intestinal and systemic disease, including severe renal damage. Upon attachment to intestinal epithelium, EHEC generates attaching and effacing (AE) lesions characterized by intimate attachment and actin rearrangement upon host cell binding. Stx produced in the gut transverses the intestinal epithelium, causing vascular damage that leads to systemic disease. Models of EHEC infection in conventional mice do not manifest key features of disease, such as AE lesions, intestinal damage, and systemic illness. In order to develop an infection model that better reflects the pathogenesis of this subset of STEC, we constructed an Stx-producing strain of Citrobacter rodentium, a murine AE pathogen that otherwise lacks Stx. Mice infected with Stx-producing C. rodentium developed AE lesions on the intestinal epithelium and Stx-dependent intestinal inflammatory damage. Further, the mice experienced lethal infection characterized by histopathological and functional kidney damage. The development of a murine model that encompasses AE lesion formation and Stx-mediated tissue damage will provide a new platform upon which to identify EHEC alterations of host epithelium that contribute to systemic disease

Genetic variants in novel pathways influence blood pressure and cardiovascular disease risk.

Blood pressure is a heritable trait influenced by several biological pathways and responsive to environmental stimuli. Over one billion people worldwide have hypertension (≥140 mm Hg systolic blood pressure or  ≥90 mm Hg diastolic blood pressure). Even small increments in blood pressure are associated with an increased risk of cardiovascular events. This genome-wide association study of systolic and diastolic blood pressure, which used a multi-stage design in 200,000 individuals of European descent, identified sixteen novel loci: six of these loci contain genes previously known or suspected to regulate blood pressure (GUCY1A3-GUCY1B3, NPR3-C5orf23, ADM, FURIN-FES, GOSR2, GNAS-EDN3); the other ten provide new clues to blood pressure physiology. A genetic risk score based on 29 genome-wide significant variants was associated with hypertension, left ventricular wall thickness, stroke and coronary artery disease, but not kidney disease or kidney function. We also observed associations with blood pressure in East Asian, South Asian and African ancestry individuals. Our findings provide new insights into the genetics and biology of blood pressure, and suggest potential novel therapeutic pathways for cardiovascular disease prevention

Zusammenarbeit von Intensivmedizin und Palliativmedizin

Hintergrund Die interdisziplinäre Zusammenarbeit zwischen Intensivmedizin und Palliativmedizin kann die Versorgungsqualität verbessern. Das Ausmaß dieser Zusammenarbeit ist aber bisher kaum untersucht. Ziel der Arbeit Es sollten die angebotenen und in Anspruch genommenen palliativmedizinischen Unterstützungsangebote auf den Intensivstationen deutscher onkologischer Spitzenzentren erfasst werden. Material und Methoden Durchgeführt wurde eine quantitativ-qualitative, deskriptive Umfrage an den 16 von der Stiftung Deutsche Krebshilfe geförderten Zentren. Die im quantitativen Teil erfragten Häufigkeiten werden als Mittelwert und Median mit den jeweiligen Streumaßen dargestellt, während die im qualitativen Teil erhobenen Triggerfaktoren mit einer Inhaltsanalyse nach Mayring ausgewertet wurden. Ergebnisse Von Juli bis August 2017 konnten Angaben aus 15 von 16 onkologischen Spitzenzentren (94 %) erfasst werden. Im Jahr 2016 wurden im Median 33 Intensivpatienten (Min. 0, Max. 100) palliativmedizinisch vorgestellt und 9 Patienten (Min. 1, Max. 30) auf eine Palliativstation verlegt. Regelmäßige intensivmedizinisch-palliativmedizinische Visiten sowie ein Screening-Tool zur Einbindung der spezialisierten Palliativmedizin sind an zwei onkologischen Spitzenzentren implementiert. Anhand von 23 genannten Triggern, die auf der Intensivstation eine palliativmedizinische Mitbehandlung ausgelöst haben, lassen sich nach qualitativer Analyse die drei Kategorien „Entscheidung und Einstellung des Teams“, „Zustand des Patienten“ und „Wunsch von Patienten und Angehörigen“ ableiten. Diskussion Trotz eines verfügbaren Angebots werden palliativmedizinische Ressourcen in den intensivmedizinischen Abteilungen der onkologischen Spitzenzentren immer noch selten genutzt. In die tägliche Routine integrierte Angebote wie Screening-Tools oder gemeinsame Visiten könnten die Ausnutzung der angebotenen palliativmedizinischen Ressourcen erhöhen und die Versorgungsqualität verbessern

BtubA-BtubB Heterodimer Is an Essential Intermediate in Protofilament Assembly

BACKGROUND:BtubA and BtubB are two tubulin-like genes found in the bacterium Prosthecobacter. Our work and a previous crystal structure suggest that BtubB corresponds to alpha-tubulin and BtubA to beta-tubulin. A 1:1 mixture of the two proteins assembles into tubulin-like protofilaments, which further aggregate into pairs and bundles. The proteins also form a BtubA/B heterodimer, which appears to be a repeating subunit in the protofilament. METHODOLOGY/PRINCIPAL FINDINGS:We have designed point mutations to disrupt the longitudinal interfaces bonding subunits into protofilaments. The mutants are in two classes, within dimers and between dimers. We have characterized one mutant of each class for BtubA and BtubB. When mixed 1:1 with a wild type partner, none of the mutants were capable of assembly. An excess of between-dimer mutants could depolymerize preformed wild type polymers, while within-dimer mutants had no activity. CONCLUSIONS:An essential first step in assembly of BtubA + BtubB is formation of a heterodimer. An excess of between-dimer mutants depolymerize wild type BtubA/B by sequestering the partner wild type subunit into inactive dimers. Within-dimer mutants cannot form dimers and have no activity